Herpes simplex virus (HSV) infections remain a common, serious problem associated with significant morbidity. After primary infection HSV establishes latency, which is not eliminated by current antiviral therapy, and latent virus is the source for viral reactivation and the recurrence of clinical disease. Despite much effort, a vaccine remains elusive. Therefore, there is a need for a new therapeutic approach that would cure latent HSV infection. Our hypothesis is that the emergence of designer rare-cutting endonucleases as powerful tools for directed genome modification offers the unique ability to selectively target, cleave, and disrupt essential viral DNA sequences within living cells. Here, we propose to use homing endonucleases (HEs) to disable HSV in latently infected neurons by targeted mutagenesis of essential viral genes, eliminating the source of viral reactivation and replication. The goal of this project is to optimize and evaluate our approach to eliminate latent HSV infection in vivo using a murine model of HSV latent infection. Moreover, our results will be directly applicable in efforts to cure varicella zoster virus, another alphaherpesvirus that like HSV establishes latency in sensory neurons. Furthermore, the data generated will be highly relevant to the development of a cure of other chronic or latent viral infections such as hepatitis B virus, HIV, or human papillomavirus. In SA1: Evaluate the efficacy in vivo of HE mutagenesis of HSV DNA in a mouse model of latent HSV infection, we will use our mouse model of latent HSV infection to test the ability of HEs to mutagenize HSV and disrupt its ability to reactivate. I SA2: Evaluate and optimize the safety in vivo of HE mutagenesis of HSV DNA in a mouse model of latent HSV infection, we will use our mouse model of latent HSV infection to evaluate and optimize the in vivo tolerability and safety of HE exposure. This project is expected to demonstrate the feasibility of our therapeutic approach directed towards the elimination of HSV pathogenesis in vivo, and to provide critical information for the development of a larger scale animal study necessary to bring this new therapeutic approach to the clinic.